The long term goals of this proposal are to use single molecule mechanical and fluorescence methods to study the mechanism by which the energy from ATP is converted to mechanical work by the molecular motor proteins, actin and myosin. Single molecule mechanical measurements have the advantage over traditional measurements with muscle fibers in that force, working stoke and kinetics of the interaction of a single cross-bridge with actin can be measured directly. A better understanding of the mechanism of contraction is an important step towards understanding and ameliorating the changes that occur in pathological conditions, resulting from defective contractility. Mutations in myosin are responsible for familial hypertropic cardiomyopathy, which is a major cause of cardiovascular death at ages under 40 and for the most common cause of congenital deaf-blind-ness in humans. Specific aims of the proposal are to: 1) determine the mechanical properties of cross-bridges by utilizing a series of ATP analogues that alter the rates of the hydrolysis mechanism; (2) utilize long-lived weakly-bound acto-myosin intermediates to characterize the prepower-stoke states; (3) combine single molecule mechanical and fluorescence methods to determine the temporal relationship between product release and force production.